U.S. patent number 5,843,927 [Application Number 08/722,488] was granted by the patent office on 1998-12-01 for 18,19-dinor-vitamin d compounds.
This patent grant is currently assigned to Wisconsin Alumni Research Foundation. Invention is credited to Hector F. DeLuca, Kato L. Perlman, Rafal R. Sicinski.
United States Patent |
5,843,927 |
DeLuca , et al. |
December 1, 1998 |
**Please see images for:
( Certificate of Correction ) ** |
18,19-dinor-vitamin D compounds
Abstract
18,19-dinor-vitamin D.sub.3 analogs in which the angular methyl
group attached to carbon 13 of the CD-ring and the exocyclic
methylene group attached to carbon 10 of the A-ring have been
removed and replaced by a hydrogen atom. The 18,19-dinor vitamin D
compounds are characterized by relatively high cell differentiation
activity, and marked intestinal calcium transport activity while
exhibiting lower activity than 1.alpha.,25-dihydroxyvitamin D.sub.3
in their ability to mobilize calcium from bone. These compounds
would be useful for the treatment of diseases where bone formation
is desired, such as osteoporosis, because of their preferential
calcemic activity, and for the treatment of psoriasis due to their
cell differentiation activity.
Inventors: |
DeLuca; Hector F. (Deerfield,
WI), Sicinski; Rafal R. (Warsaw, PL), Perlman;
Kato L. (Madison, WI) |
Assignee: |
Wisconsin Alumni Research
Foundation (Madison, WI)
|
Family
ID: |
23343562 |
Appl.
No.: |
08/722,488 |
Filed: |
September 27, 1996 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
Issue Date |
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342855 |
Nov 21, 1994 |
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Current U.S.
Class: |
514/167;
552/653 |
Current CPC
Class: |
C07F
7/1804 (20130101); A61P 35/00 (20180101); A61P
43/00 (20180101); A61P 17/00 (20180101); A61P
3/02 (20180101); A61P 19/10 (20180101); C07C
401/00 (20130101) |
Current International
Class: |
C07C
401/00 (20060101); C07F 7/00 (20060101); C07F
7/18 (20060101); A61K 031/59 (); C07C 401/00 () |
Field of
Search: |
;552/653 ;514/167 |
References Cited
[Referenced By]
U.S. Patent Documents
Foreign Patent Documents
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A0516410 |
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Dec 1992 |
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EP |
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A0582481 |
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Feb 1994 |
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EP |
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A0664287 |
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Jul 1995 |
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EP |
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WOA9501960 |
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Jan 1995 |
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WO |
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Other References
Biorganic & Medicinal Chemistry Letters, vol. 3, No. 9, pp.
1855-1858, 1993, "Synthesis and Biological Evaluation of
18-Substituted Analogs of 1.alpha.,25-Dihydroxyvitamin D.sub.3 ",
Nilsson et al. .
J. Org. Chem., vol. 57, No. 11, pp. 3214-3217, 1992,
"18-Substituted Derivatives of Vitamin D:
18-Acetoxy-1.alpha.,25-Dihydroxyvitamin D.sub.3 and Related
Analogues", Maynard et al..
|
Primary Examiner: Dees; Jose G.
Assistant Examiner: Pryor; Alton
Attorney, Agent or Firm: Andrus, Sceales, Starke &
Sawall
Government Interests
This invention was made with United States Government support
awarded by the National Institutes of Health (NIH), Grant No.
DK-14881. The United States Government has certain rights in this
invention.
Parent Case Text
This application is a continuation of application Ser. No.
08/342,855 filed Nov. 21, 1994, now abandoned.
Claims
We claim:
1. A compound having the formula: ##STR9## where X.sup.1 and
X.sup.2, which may be the same or different, are each selected from
hydrogen and a hydroxy protecting group, and where the group R is
represented by the structure: ##STR10## where the stereochemical
center at carbon 20 may have the R or S configuration, and where Z
is selected from Y, --OY, --CH.sub.2 OY, --C.ident.CY and --CH=CHY,
where the double bond may have the cis or trans geometry, and where
Y is selected from hydrogen, methyl, --CR.sup.5 O and a radical of
the structure: ##STR11## where m and n, independently, represent
the integers from 0 to 5, where R.sup.1 is selected from hydrogen,
deuterium, hydroxy, protected hydroxy, fluoro, trifluoromethyl, and
C.sub.1-5 -alkyl, which may be straight chain or branched and,
optionally, bear a hydroxy or protected-hydroxy substituent, and
where each of R.sup.2, R.sup.3, and R.sup.4, independently, is
selected from deuterium, deuteroalkyl, hydrogen, fluoro,
trifluoromethyl and C.sub.1-5 alkyl, which may be straight-chain or
branched, and optionally, bear a hydroxy or protected-hydroxy
substituent, and where R.sup.1 and R.sup.2, taken together,
represent an oxo group, or an alkylidene group, =CR.sup.2 R.sup.3,
or the group --(CH.sub.2).sub.p --, where p is an integer from 2 to
5, and where R.sup.3 and R.sup.4, taken together, represent an oxo
group, or the group --(CH.sub.2).sub.q --, where q is an integer
from 2 to 5, and where R.sup.5 represents hydrogen, hydroxy,
protected hydroxy, or C.sub.1-5 alkyl.
2. A pharmaceutical composition containing at least one compound as
claimed in claim 1 together with a pharmaceutically acceptable
excipient.
3. The pharmaceutical composition of claim 2 containing
18,19-dinor-1.alpha.,25-dihydroxvvitamin D.sub.3 in an amount from
about 0.1 .mu.g to about 50 .mu.g.
4. 18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3.
5. 18,19-dinor-1.alpha.-hydroxyvitamin D.sub.3.
Description
BACKGROUND OF THE INVENTION
The natural hormone, 1.alpha.,25-dihydroxyvitamin D.sub.3 and its
analog in ergosterol series, i.e. 1.alpha.,25-dihydroxyvitamin
D.sub.2 are known to be highly potent regulators of calcium
homeostasis in animals and humans, and more recently their activity
in cellular differentiation has been established, V. Ostrem et al,
Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Many structural
analogs of these metabolites have been prepared and tested,
including 1.alpha.-hydroxyvitamin D.sub.3, 1.alpha.-hydroxyvitamin
D.sub.2, various side chain homologated vitamins and fluorinated
analogs. Some of these compounds exhibit an interesting separation
of activities in cell differentiation and calcium regulation. This
difference in activity may be useful in the treatment of a variety
of diseases as renal osteodystrophy, vitamin D-resistant rickets,
osteoporosis, psoriasis, and certain malignancies.
Recently, a new class of vitamin D analogs has been discovered,
i.e. so called 19-nor-vitamin D compounds, which are characterized
by the replacement of the ring A exocyclic methylene group (carbon
19), typical of the vitamin D system, by hydrogen atoms. Biological
testing of such 19-nor-analogs (e.g.
1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3) revealed a selective
activity profile with high potency in inducing cellular
differentiation, with very low calcium mobilizing activity. Thus,
these compounds are potentially useful as therapeutic agents for
the treatment of malignancies, or the treatment of various skin
disorders. Two different methods of synthesis of such
19-nor-vitamin D analogs have been described (Perlman et al.
Tetrahedron Letters 31, 1823 (1990); Perlman et al. Tetrahedron
Letters 32, 7663 (1991), and DeLuca et al. U.S. Pat. No.
5,086,191).
In a continuing effort to explore the 19-nor class of
pharmacologically important vitamin D compounds, their analogs
lacking the C-18 angular methyl group, i.e. 18,19-dinor-vitamin D
compounds have now been synthesized and tested.
DISCLOSURE OF THE INVENTION
A class of 1.alpha.-hydroxylated vitamin D compounds not known
heretofore are the 18,19-dinor-analogs, i.e. compounds in which the
C-18 angular methyl substituent (carbon 18) normally attached to
carbon 13 of the CD-ring and the C-19 exocyclic methylene group
(carbon 19) normally attached to carbon 10 of the A-ring which are
typical of all vitamin D systems have been removed and replaced by
hydrogen atoms. Structurally these novel analogs are characterized
by the general formula I shown below: ##STR1## where X.sup.1 and
X.sup.2, which may be the same or different, are each selected from
hydrogen and a hydroxy protecting group, and where the group R
represents any of the typical side chains known for vitamin D type
compounds.
More specifically R can represent a saturated or unsaturated
hydrocarbon radical of 1 to 35 carbons, that may be straight-chain,
branched or cyclic and that may contain one or more additional
substituents, such as hydroxy- or protected-hydroxy groups, fluoro,
carbonyl, ester, epoxy, amino or other heteroatomic groups.
Preferred side chains of this type are represented by the structure
below. ##STR2## where the stereochemical center (corresponding to
C-20 in, steroid numbering) may have the R or S configuration,
(i.e. either the natural configuration about carbon 20 or the
20-epi configuration), and where Z is selected from Y, --OY,
--CH.sub.2 OY, --C=CY and --CH=CHY, where the double bond may have
the cis or trans geometry, and where Y is selected from hydrogen,
methyl, --CR.sup.5 O and a radical of the structure: ##STR3## where
m and n, independently, represent the integers from 0 to 5, where
R.sup.1 is selected from hydrogen, deuterium, hydroxy, protected
hydroxy, fluoro, trifluoromethyl, and C.sub.1-5 -alkyl, which may
be straight chain or branched and, optionally, bear a hydroxy or
protected-hydroxy substituent, and where each of R.sup.2, R.sup.3,
and R.sup.4, independently, is selected from deuterium,
deuteroalkyl, hydrogen, fluoro, trifluoromethyl and C.sub.1-5
alkyl, which may be straight-chain or branched, and optionally,
bear a hydroxy or protected-hydroxy substituent, and where R.sup.1
and R.sup.2, taken together, represent an oxo group, or an
alkylidene group, =CR.sup.2 R.sup.3, or the group
--(CH.sub.2).sub.p --, where p is an integer from 2 to 5, and where
R.sup.3 and R.sup.4, taken together, represent an oxo group, or the
group --(CH.sub.2).sub.q --, where q is an integer from 2 to 5, and
where R.sup.5 represents hydrogen, hydroxy, protected hydroxy, or
C.sub.1-5 alkyl and wherein any of the CH-groups at positions 20,
22, or 23 in the side chain may be replaced by a nitrogen atom, or
where any of the groups --CH(CH.sub.3)--, --CH(R.sup.3)--, or
--CH(R.sup.2)-- at positions 20, 22, and 23, respectively, may be
replaced by an oxygen or sulfur atom.
Specific important examples of side chains are the structures
represented by formulas (a), (b), (c), (d) and (e) below. i.e. the
side chain as it occurs in 25-hydroxyvitamin D.sub.3 (a); vitamin
D.sub.3 (b); 25-hydroxyvitamin D.sub.2 (c); vitamin D.sub.2 (d);
and the C-24 epimer of 25-hydroxyvitamin D.sub.2 (e). ##STR4##
The above novel compounds exhibit a desired, and highly
advantageous, pattern of biological activity. These compounds are
characterized by a marked intestinal calcium transport activity, as
compared to that of 1.alpha., 25-dihydroxyvitamin D.sub.3, while
exhibiting lower activity than 1.alpha.,25-dihydroxyvitamin D.sub.3
in their ability to mobilize calcium from bone. Hence, these
compounds are highly specific in their calcemic activity. Their
preferential activity on intestinal calcium transport and reduced
calcium mobilizing activity in bone allows the in vivo
administration of these compounds for the treatment of metabolic
bone diseases where bone loss is a major concern. Because of their
preferential calcemic activity, these compounds would be preferred
therapeutic agents for the treatment of diseases where bone
formation is desired, such as osteoporosis, osteomalacia and renal
osteodystrophy. The treatment may be transdermal, oral or
parenteral. The compounds may be present in a composition in an
amount from about 0.1 .mu.g/gm to about 50 .mu.g/gm of the
composition, and may be administered in dosages of from about 0.1
.mu./day to about 50 .mu.g/day.
The above compounds are also characterized by high cell
differentiation activity. Thus, these compounds also provide
therapeutic agents for the treatment of psoriasis. The compounds
may be present in a composition to treat psoriasis in an amount
from about 0.01 .mu.g/gm to about 100 .mu.g/gm of the composition,
and may be administered topically, orally or parenterally in
dosages of from about 0.01 .mu.g/day to about 100 .mu.g/day.
This invention also provides novel intermediate compounds formed
during the synthesis of the end products.
BRIEF DESCRIPTION OF THE DRAWINGS
FIG. 1 is a graph illustrating the percent HL-60 cell
differentiation as a function of the concentration of
18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3,
19-nor-1.alpha.,25-dihydroxyvitamin D.sub.3 and
1.alpha.,25-dihydroxyvitamin D.sub.3 ; and
FIG. 2 is a graph illustrating the relative activity of
18-nor-1.alpha.,25-dihydroxyvitamin D.sub.3,
19-nor-1.alpha.,25-dihydroxyvitamin D.sub.3,
18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3, and
1.alpha.,25-dihydroxyvitamin D.sub.3 in binding to the
1,25-dihydroxyvitamin D pig intestinal nuclear receptor.
DETAILED DESCRIPTION OF THE INVENTION
As used in the description and in the claims, the term
"hydroxy-protecting group" signifies any group commonly used for
the temporary protection of hydroxy functions, such as for example,
alkoxycarbonyl, acyl, alkylsilyl or alkylarylsilyl groups
(hereinafter referred to simply as "silyl" groups), and alkoxyalkyl
groups. Alkoxycarbonyl protecting groups are groupings such as
methoxycarbonyl, ethoxycarbonyl, propoxycarbonyl,
isopropoxycarbonyl, butoxycarbonyl, isobutoxycarbonyl,
tert-butoxycarbonyl, benzyloxycarbonyl or allyloxycarbonyl. The
term "acyl" signifies an alkanoyl group of 1 to 6 carbons, in all
of its isomeric forms, or a carboxyalkanoyl group of 1 to 6
carbons, such as an oxalyl, malonyl, succinyl, glutaryl group, or
an aromatic acyl group such as benzoyl, or a halo, nitro or alkyl
substituted benzoyl group. The word "alkyl" as used in the
description or the claims, denotes a straight-chain or branched
alkyl radical of 1 to 10 carbons, in all its isomeric forms.
Alkoxyalkyl protecting groups are groupings such a methoxymethyl,
ethoxymethyl, methoxyethoxymethyl, or tetrahydrofuranyl and
tetrahydropyranyl. Preferred silyl-protecting groups are
trimethylsilyl, triethylsilyl, t-butyldimethylsilyl,
dibutylmethylsilyl, diphenylmethylsilyl, phenyldimethylsilyl,
diphenyl-t-butylsilyl and analogous alkylated silyl radicals.
A "protected hydroxy" group is a hydroxy group protected by any
group commonly used for the temporary or permanent protection of
hydroxy functions, e.g. the silyl, alkoxyalkyl, acyl or
alkoxycarbonyl groups, as previously defined. The terms
"hydroxyalkyl", "deuteroalkyl" and "fluoroalkyl" refer to an alkyl
radical substituted by one or more hydroxy, deuterium or fluoro
groups respectively.
The preparation of 1.alpha.-hydroxy-18,19-dinor-vitamin D.
compounds having the basic structure I can be accomplished by a
common general method, i.e. the condensation of the ring A synthon
II with a bicyclic Windaus-Grundmann type ketone III: ##STR5## In
the structures II and III, groups X.sup.1, X.sup.2 and R represent
groups defined above; X.sup.1 and X.sup.2 are preferably
hydroxy-protecting groups, it being also understood that any
functionalities in R that might be sensitive, or that interfere
with the condensation reaction, be suitable protected as is
well-known in the art. Compounds of the general structure III,
where Y is --POPh.sub.2, PO(Alkyl).sub.2, or --SO.sub.2 Ar, or
--Si(Alkyl).sub.3 can be prepared by described method (DeLuca et
al., Eur. Pat. Appl. EP 0 516 410 A2). Phosphine oxide of structure
II, with tert-butyldimethylsilyl groups as X.sup.1 and X.sup.2, is
the known compound, [Perlman et al., Tetrahedron Letters 32, 7663
(1991)] which can be succesfully used for the above condensation.
The process shown above represents an application of the convergent
synthesis concept, which has been applied effectively for the
preparation of vitamin D compounds [e.g. Lythgoe et al., J. Chem.
Soc. Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev. 9, 449
(1983); Toh et al., J. Org. Chem. 48, 1414 (1983); Baggiolini et
al., J. Org. Chem. 51, 3098 (1986); Sardina et al., J. Org. Chem.
51, 1264 (1986); J. Org. Chem. 51, 1269 (1986)].
For the preparation of the 18-nor CD ketones of general structure
III, a new synthetic route has been developed, based on the
Windaus-Grundmann type ketones of the general structure IV as
starting materials. Required CD-ring ketones IV are known, or can
be prepared by known methods. Specific important examples of such
known bicyclic ketones are the structures with the side chains (a),
(b), (c) and (d) described above, i.e. 25-hydroxy Grundmann's
ketone (e) [Baggiolini et al., J. Org. Chem, 51, 3098 (1986)];
Grundmann's ketone (f) [Inhoffen et al., Chem. Ber. 90, 664
(1957)]; 25-hydroxy Windaus ketone (g) [Baggiolini et al., J. Org.
Chem., 51, 3098 (1986)] and Windaus ketone (h) [Windaus et al.,
Ann., 524, 297 (1936)]: ##STR6##
The overall process of transformation of the starting bicyclic
ketones IV into their 18-nor analogs III, in general form, is
summarized by the reaction scheme below: ##STR7## As shown in this
scheme, first step of the synthesis comprises the reduction of the
8-keto group in IV to the axial 8.beta.-hydroxy CD-fragment V
(X.sup.3 =H). Such stereoselective reduction process is well known
and can be easily accomplished using, for example, LiAlH.sub.4 or
NaBH.sub.4. It is understood that hydroxy groups in the side chain
R of ketone IV, if present, should be approppriately protected
before the reduction process, and the protecting groups selected
are both compatible with subsequent chemical transformations, and
also removable, if desired. Suitable are, for example, alkylsilyl-
and arylsilyl groups or alkoxyalkyl groups.
The axial orientation of the C-8 hydroxy group in V (X.sup.3 =H),
being sterically fixed in the trans-hydrindane system, in close
proximity to the angular methyl group at C-13, is crucial for the
successful intramolecular free radical reaction leading to
18-functionalized compounds. It has been established that
efficiency of the abstraction of a hydrogen atom from the angular
methyl group in steroids strongly depends on the distance of the
oxy radical from the hydrogen atoms of the angular methyl groups.
The rate of hydrogen abstraction reaches a maximum at internuclear
distances between oxygen and the methyl carbon of 2.5-2.7 A and
decreases rapidly at distances over 3 .ANG.. Our molecular modeling
studies show that in the case of 8.beta.-alcohols V (X.sup.3 =H)
the distance C(18)-O is smaller than 3 .ANG. (usually ca. 2.96
.ANG.) and, therefore, these compounds fulfill all requirements for
successful functionalization at C-18. As a method of angular methyl
group functionalization a photolysis of nitrites (Barton reaction)
has been chosen. Thus, alcohols of general structure V (X.sup.3 =H)
are converted into the corresponding nitrites V (X.sup.3 =NO) by
one of the existing methods, including treatment with nitrosyl
chloride in pyridine and trans-esterification with tert-butyl
nitrite or isopentyl nitrite. The former method has a more general
applicability but requires the use of expensive gaseous nitrosyl
chloride. The latter, nitrosyl exchange method, can be recomended
due to its simplicity. The next step of the synthesis consisted of
the photolysis of V (X.sup.3 =NO) resulting in the intramolecular
exchange of the NO of the nitrite residue with hydrogen atom
attached to C-18. The C-nitroso compound VI thus formed rearranges
to hydroxy oxime VII (X.sup.4 =H) either spontaneously or by
heating in a solvent such as 2-propanol. Nitrite V (X.sup.3 =NO)
photolysis can be in general performed under oxygen-free atmosphere
in an irradiation apparatus with a water-cooled central sleeve into
which the mercury lamp equipped with pyrex filter is introduced and
efficient cooling is used to keep the temperature of the irradiated
solution between 0.degree. and 10.degree. C. The drop in yield, due
to competing intermolecular hydrogen abstraction reactions
(regenerating the starting alcohol), can be suppressed by using
solvents which do not contain easily abstractable hydrogen atoms,
e.g. benzene. Although 18-nitroso compounds of general structure VI
usually isomerize rapidly to the 18-oximes VII (X.sup.4 =H), it is
recommended that rearrangement be completed by brief treatment of
the crude irradiation product in boiling 2-propanol.
The subsequent steps of the process comprise the transformation of
8.beta.-hydroxy oxime VII (X.sup.4 =H) into the 8.beta.-hydroxy
nitrile VIII (X.sup.5 =H). This conversion can be easily achieved
by the thermal elimination of the elements of acetic acid from the
acetyl derivative VII (X.sup.4 =Ac) folowed by hydrolysis of
8.beta.-acetoxy group in the resulting acetoxy nitrile VIII
(X.sup.5 =Ac). The transformation of hydroxy oxime VII (X.sup.4 =H)
to VIII (X.sup.5 =Ac) can be done in two steps: acetylation of VII
(X.sup.4 =H) under standard conditions (acetic anhydride in
pyridine) to diacetate VII (X.sup.4 =Ac) and subsequent thermal
reaction (pyrolysis) of the latter resulting in the elimination of
acetic acid molecule ,from the acetoxyimino group and formation of
the nitrile VIII (X.sup.5 =Ac). Alternatively, the conversion of
VII (X.sup.4 =H) to VIII (X.sup.5 =Ac) can be much easier
accomplished by heating the oxime in acetic anhydride (addition of
sodium or potassium acetate is sometimes helpful).
The hydrolysis of 8.beta.-acetoxy group in the nitrile VIII
(X.sup.5 =Ac) producing the corresponding alcohol VIII (X.sup.5 =H)
can be performed under standard basic conditions. This process is
desired in view of the following chemical transformation, i.e.
reductive removal of the C-13 cyano group. Conditions required for
such decyanation process could otherwise cause the reduction of the
8-acetoxy group to the corresponding alkane (8-unsubstituted
derivative). 8.beta.-Hydroxy group in VIII (X.sup.5 =H) can be
protected as alkylsilyl-, arylsilyl or alkoxyalkyl ether during the
decyanation process, if desired. It is understood, however, that
such protecting group has to be selectively deprotectable (in the
presence of other protected hydroxy groups in R, if any) at the
next stage of the synthesis. Several methods for the reductive
decyanation of VIII (X.sup.5 =H) are available, the most important
being dissolving metal reductions. Thus, for example, VIII (X.sup.5
=H) can be transformed into 18-nor derivative IX by reaction with
potassium metal in hexamethylphosphoric triamide and tert-butanol
or using potassium metal/dicyclohexano-18-crown-6/toluene
system.
The following synthetic step comprises the oxidation of
18-nor-8.beta.-alcohol IX to the desired 8-keto compound III.
Several oxidation methods can be used providing they do not cause
epimerization at C-14 in the formed product. Methods recommended
for their ability to preserve a chiral center next to 8-keto group
include oxidation with CrO.sub.3 -pyridine, SO.sub.3 -Me.sub.2 SO
and PDC reagents. Keto compound III can be directly used in the
next Wittig-Horner reaction giving 18,19-dinor-vitamin D
derivatives I or, before the coupling step, it can be transformed
to another compound with different side chain R. In the case where
R is a saturated side chain, for example cholestane side chain (b)
(18-nor Grundmann's ketone), there is a possibility to perform
selective hydroxylation of the unhindered tertiary carbon atom
(C-25 in the case of cholestane side chain) using ruthenium
tetroxide [Kiegiel et al., Tetrahedron Letters 32, 6057 (1991)] or
dioxirane [Bovicelli et al., J. Org. Chem., 57, 5052 (1992)]
oxidation methods. If desired, 8.beta.-alcohol IX can be subjected
to side chain hydroxylation process because, under the reaction
conditions, rapid oxidation of a secondary hydroxy group at C-8
takes place.
The condensation reaction is advantageously conducted by treating
the A ring-unit of general structure II, dissolved in an organic
solvent, with a strong base (e.g. an alkali metal hydride, alkyl-
or aryl lithium, or a lithium alkylamide reagent), so as to
generate the anion of II, and then allowing this anion to react
with 18-nor-ketone III, so as to achieve condensation to the
18,19-dinor-vitamin D analog I, either directly, or via
intermediates (e.g. in the case of condensation with compound II
where Y=SO.sub.2 Ar) transformable to I according to known
procedures. Any hydroxy-protecting groups (i.e. protecting groups
X.sup.1 and X.sup.2 and/or hydroxy-protecting groups that may be
present in the side chain R) can then be removed by appropriate
hydrolytic or reductive procedures known in the art to obtain the
free hydroxy-vitamin analog, structure I, where X.sup.1 and X.sup.2
represent hydrogen.
Synthesis of 1.alpha.,25-dihydroxy-18,19-dinor-vitamin D.sub.3
EXAMPLE 1
Preparation of des-A,B-cholestan-8S-yl nitrite (4)
(Scheme 1)
A solution of Grundmann's ketone 2 [(2.70 g, 10.2 mmol; obtained by
ozonolysis of commercial vitamin D.sub.3 (1)] in anhydrous ether
(90 mL) at 0.degree. C. was added to a slurry of LiAlH.sub.4 (3.89
g, 102.5 mmol) in anhydrous ether (270 mL). The reaction mixture
was stirred at 0.degree. C. for 1 h, and ethyl acetate (27 mL)
followed by cold 10% H.sub.2 SO.sub.4 (100 mL) was used to destroy
the unreacted LiAlH.sub.4 and complete the hydrolysis. The
resulting mixture was extracted with ether, the combined extracts
were washed with water and brine, dried (Na.sub.2 SO.sub.4) and
evaporated. The product was purified by flash chromatography on
silica. Elution with 10% ethyl acetate in hexane gave the known
8.beta.-alcohol 3 as a colorless oil (2.42 g, 89%): .sup.1 H NMR
(CDCl.sub.3, 500 MHz) .delta.0.865 (6H, br d, J.about.6 Hz, 26- and
27-H.sub.3), 0.891 (3H, d, J=6.4 Hz, 21-H.sub.3), 0.929 (3H, s,
18-H.sub.3), 4.07 (1H, m, w/2=10 Hz, 8.alpha.-H) ; MS m/z (relative
intensity) 266 (M.sup.+, 9), 251 (3), 207 (12), 164 (19), 111 (61),
91 (100).
A solution of alcohol 3 (533 mg, 2 mmol) in chloroform (10 mL) was
treated with tert-butyl nitrite (2.2 mL) and stirred at room
temperature in the dark for 40 min. Benzene (20 mL) was added and
the solvents were rapidly evaporated under vacuum (temperature of
water bath 40.degree. C.). During evaporation of solvents and
further high-vacuum drying the nitrite was protected from light.
The oily product contained traces of starting alcohol 3 but it was
suitable for the subsequent reaction. The nitrite 4 possessed the
following spectral characteristics: IR (CHCl.sub.3) 1632 (nitrite)
cm.sup.1 ; .sup.1 H NMR (CDCl.sub.3, 500 MHz) 67 0.767 (3H, s,
18-H.sub.3), 0.862 (6H, br d, J=6.2 Hz, 26- and 27-H.sub.3), 0.901
(3H, d, J=7.0 Hz, 21- H.sub.3), 5.76 (1H, narr m, 8.alpha.-H).
EXAMPLE 2
Synthesis of 18-(hydroxyimino)-des-A,B-cholestan-8.beta.-ol (6)
The crude nitrite ester 4 obtained from 2 mmol of 8.beta.-alcohol 3
(see Example 1) was dissolved in anhydrous benzene (140 mL) and
irradiated, in the apparatus consisting of a Pyrex vessel and a
water-cooled Vycor immersion well, with Hanovia high pressure
mercury arc lamp equipped with a Pyrex filter. Slow stream of argon
was passed into the vessel and the temperature of the solution was
maintained at 10.degree. C. After 1 h 40 min of the irradiation TLC
showed only traces of unreacted nitrite. The reaction mixture was
allowed to stand overnight at room temperature (in order to
accomplish an isomerization of the intermediate 19-nitroso compound
5 to the oxime), benzene was evaporated under vacuum and the oily
residue was subjected to flash chromatography. Elution with 30%
ethyl acetate in hexane afforded pure oxime 6 (270 mg, 46% from
8.beta.-alcohol 3) as a colorless oil: IR (CHCl.sub.3) 3590, 3240,
3140 (OH) cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3) .delta. 0.865 (6H,
d, J=6.1 Hz, 26- and 27-H.sub.3), 0.994 (3H, d, J=6.7 Hz, 21-
H.sub.3), 4.04 (1H, m, w/2=9 Hz, 8.alpha.-H), 6.29 (1H, br s, OH),
7.36 (1H, s, 18-H), 10.38 (1H, br s, OH); MS m/z (relative
intensity) 295 (M.sup.+, 16), 278 (87), 260 (68), 245 (33), 183
(100); exact mass calcd for C.sub.18 H.sub.33 O.sub.2 N 295.2511,
found 295.2514.
EXAMPLE 3
Conversion of oxime 6 into
8.beta.-acetoxy-des-A,B-cholestane-18-nitrile (8)
(a) A solution of the oxime 6 (120 mg, 0.41 mmol) in acetic
anhydride (5 mL) was refluxed for 1.5 h. The reaction mixture was
cooled, poured carefully on ice and extracted with benzene.
Extracts were combined, washed with water, NaHCO.sub.3 and brine,
dried (Na.sub.2 SO.sub.4) and evaporated. The oily residue was
purified by flash chromatography using 10% ethyl acetate in hexane.
Pure acetoxy nitrile 8 (112 mg, 86%) was obtained as a colorless
oil: IR (CHCl.sub.3) 2220 (nitrile), 1720 and 1240 (acetate)
cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3) .delta.0.864 (6H, d, J=6.2
Hz, 26- and 27-H.sub.3), 1.032 (3H, d, J=6.5 Hz, 21-H.sub.3), 2.13
(3H, s, OAc), 5.20 (1H, m, w/2=8 Hz, 8.alpha.-H); MS m/z (relative
intensity) 319 (M.sup.-, 56), 304 (18), 277 (89), 259 (100), 244
(64); exact mass calcd for C.sub.20 H.sub.33 O.sub.2 N 319.2511,
found 319.2506.
(b) Hydroxy oxime 6 (120 mg, 0.41 mmol) was heated with acetic
anhydride (0.3 mL) and pyridine (0.5 mL) for 36 h at 60.degree. C.
The reaction mixture was cooled, poured on ice and extracted with
benzene. Extracts were combined, washed with water, NaHCO.sub.3 and
brine, dried (Na.sub.2 SO.sub.4) and evaporated. The oily residue
was purified by flash chromatography using 10% ethyl acetate in
hexane. Pure acetoxy nitrile 8 (109 mg, 84%) was obtained as a
colorless oil.
Monitoring of the reaction mixture with TLC showed a presence of a
spot corresponding to diacetate 7.
EXAMPLE 4
Hydrolysis of the acetoxy nitrile 8 to
8.beta.-hydroxy-des-A,B-cholestane-18-nitrile (9)
Acetoxy nitrile 8 (210 mg, 0.66 mmol) was treated with 10%
methanolic KOH (10 mL) at 50.degree. C. for 1.5 h. After
concentration under vacuum the reaction mixture was poured into
water and extracted with benzene and ether. The organic extracts
were combined, washed with brine, dried (Na.sub.2 SO.sub.4) and
evaporated. The residue was redissolved in hexane/ethyl acetate
(7:3) and the solution passed through a silica gel Sep-Pak
cartridge. Evaporation of solvents gave pure hydroxy nitrile 9 (175
mg, 96%) as an oil: IR (CHCl.sub.3) 3600 (OH), 2220 (nitrile)
cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3) .delta. 0.868 (6H, d, J=6.0
Hz, 26- and 27-H.sub.3), 1.032 (3H, d, J=7.1 Hz, 21-H.sub.3), 4.10
(1H, m, w/2=10 Hz, 8.alpha.-H); MS m/z (relative intensity) 277
(M.sup.+, 37), 262 (28), 244 (18), 234 (26), 220 (32), 206 (87),
121 (100); exact mass calcd for C.sub.18 H.sub.31 ON 277.2406,
found 277.2406.
EXAMPLE 5
Reductive decyanation of hydroxy nitrile 9 to
des-A,B-18-norcholestan-8.beta.-ol (10)
(a) To a stirred mixture of potassium (55 mg, 1.4 mmol) in
hexamethylphosphoric triamide (HMPA, 170 .mu.L) and ether (420
.mu.L) a solution of the hydroxy nitrile 9 (55 mg, 0.2 mmol) in
tert-butanol (50 .mu.L) and ether (200 .mu.L) was added dropwise at
0.degree. C. under argon. Cooling bath was removed and the
brown-yellow solution was stirred at room temperature for 5 h under
argon. Unreacted potassium was removed, the mixture was diluted
with benzene, few drops of 2-propanol were added and water. The
organic phase was washed with water, dried (Na.sub.2 SO.sub.4) and
evaporated. The residue was purified by flash chromatography.
Elution with 10% ethyl acetate in hexane gave pure alcohol 10 (38
mg, 76%) as a colorless oil: IR (CHCl.sub.3) 3630 and 3470 (OH)
cm.sup.-1 ; .sup.1 H NMR (CDCl.sub.3) .delta. 0.863 and 0.868 (3H
and 3H, each d, J=6.3 Hz, 26- and 27-H.sub.3), 0.881 (3H, d, J=6.5
Hz, 21-H.sub.3), 4.05 (1H, m, w/2=8 Hz, 8.alpha.-H) ; .sup.1 H NMR
(C.sub.6 D.sub.6) .delta. 0.901 and 0.907 (3H and 3H, each d, J=6.2
Hz, 26- and 27-H.sub.3), 0.945 (3H, d, J=6.5 Hz, 21-H.sub.3), 3.80
(1H, m, w/2=8 Hz, 8.alpha.-H); .sup.13 C NMR (CDCl.sub.3) .delta.
18.1 (q), 20.3 (t), 22.5 (q), 22.7 (q), 24.8 (t), 25.4 (t), 25.6
(t), 27.9 (d), 31.7 (t), 33.5 (t+t), 35.1 (d), 39.3 (t), 39.6 (d),
49.8 (d), 50.7 (d), 67.9 (d); MS m/z (relative intensity) 252
(M.sup.+, 1), 234 (3), 219 (2), 121 (100); exact mass calcd for
C.sub.17 H.sub.32 O 252.2453, found 252.2470.
(b) A lump (ca. 1/4 cm.sup.3) of potassium metal was added to a
solution of hydroxy nitrile 9 (55 mg, 0.2 mmol) and
dicyclohexano-18-crown-6 (111 mg, 0.3 mmol) in anhydrous toluene (8
mL). The mixture was stirred under argon at room temperature for 10
h, unreacted potassium was removed, few drops of 2-propanol were
added and water. The organic phase was washed with water, dried
(Na.sub.2 SO.sub.4) and evaporated. The residue was subjected to
flash chromatography. Elution with 10% ethyl acetate in hexane gave
alcohol 10 (30 mg) which was subsequently purified by HPLC (10
mm.times.25 cm Zorbax-Sil column, 4 mL/min) using hexane/ethyl
acetate (9:1) solvent system. Pure compound 10 (25 mg, 50%) was
eluted at R.sub.v 44 mL as a colorless oil.
EXAMPLE 6
Oxidation of alcohol 10 to des-A,B-18-norcholestan-8-one (11) and
25-hydroxy-des-A,B-18-norcholestan-8-one (12)
(a) To a solution of alcohol 10 (5 mg, 20 .mu.mol) in CH.sub.2
Cl.sub.2 (2 mL) containing a catalytic amount of pyridinium
p-toluenesulfonate (PPTS) was added pyridinium dichromate (PDC, 25
mg, 66 .mu.mol) at 0.degree. C. with stirring. After 10 min the
cooling bath was removed and the mixture was stirred at room
temperature for 5 h. The brown mixture was diluted with ether and
filtered through a silica Sep-Pak that was washed with hexane/ethyl
acetate (1:1). Evaporation of the solvents gave a crude ketone 11
which was further purified by HPLC (10 mm.times.25 cm Zorbax-Sil
column, 4 mL/min) using hexane/ethyl acetate (9:1) solvent system.
Analytically pure compound 11 (4 mg, 80%) was eluted at R.sub.v 29
mL (Grundmann's ketone 2 was eluted at R.sub.v 31 mL in the same
system): [.alpha.].sup.22.sub.D +16.2.degree. (c 0.31, CHCl.sub.3);
CD .DELTA..epsilon. (.lambda..sub.max): -0.76 (311), -1.32 (301),
-1.34 (294), -0.92 (282), -1.33 (190); .sup.1 H NMR (CDCl.sub.3)
.delta.0.866 (6H, d, J=6.9 Hz, 26- and 27-H.sub.3), 0.889 (3H, d,
J=6.9 Hz, 21-H.sub.3); .sup.13 C NMR (CDCl.sub.3) .delta. 18.0 (q),
21.5 (t), 22.5 (q), 22.7 (q), 25.4 (t+t), 27.8 (t), 27.9 (d), 30.6
(t), 33.2 (t), 34.8 (d), 39.3 (t), 41.5 (t), 50.8 (d), 50.9 (d),
58.3 (d), 212.0 (s); MS m/z (relative intensity) 250 (M.sup.+, 80),
207 (44), 137 (100); exact mass calcd for C.sub.17 H.sub.30 O
250.2297, found 250.2289.
(b) To the stirred solution of ruthenium (III) chloride hydrate
(11.5 mg, 0.06 mmol) and NaIO.sub.4 (263 mg, 1.23 mmol) in water
(1.0 mL), a solution of alcohol 10 (85 mg, 0.34 mmol) in CCl.sub.1
-CH.sub.3 CN (1:1, 1.5 mL) was added. The mixture was vigorously
stirred for 72 h at room temperature. Few drops of 2-propanol were
added, the mixture was poured into water and extracted with
CCl.sub.4 /CHCl.sub.3 solvent system. The combined organic extracts
were washed with water, dried (Na.sub.2 SO.sub.4) and evaporated to
give an oily residue which was subjected to flash chromatography.
Elution with 20% ethyl acetate in hexane gave 8-ketone 11 (16 mg,
19%). Subsequent elution with 40% ethyl acetate in hexane afforded
impure 25-hydroxy ketone 12 (20 mg) which was subjected to HPLC (10
mm.times.25 cm Zorbax-Sil column, 4 mL/min) using hexane/ethyl
acetate (6:4) solvent system. Analytically pure compound 12 (12.7
mg, 14%;) was eluted at R.sub.v 51 mL (25-hydroxy Grundmann's
ketone was eluted at R.sub.v 50 mL in the same system) as an oil
crystallizing on standing in the refrigerator: .sup.1 H NMR
(CDCl.sub.3) .delta. 0.908 (3H, d, J=6.5 Hz, 21-H.sub.3), 1.216
(6H, s, 26- and 27-H.sub.3); .sup.13 C NMR (CDCl.sub.3) 67 18.0
(q), 21.5 (t), 22.3 (t), 25.4 (t), 27.8 (t), 29.3 (q+q), 30.6 (t),
33.5 (t), 34.8 (d), 41.5 (t), 44.2 (t), 50.8 (d), 50.9 (d), 58.3
(d), 71.0 (s), 211.9 (s); MS m/z (relative intensity) 266 (M.sup.+,
<1), 251 (6), 248 (60), 233 (16), 137 (100); exact mass calcd
for C.sub.17 H.sub.30 O.sub.2 266.2246, found 266.2257.
EXAMPLE 7
Silylation of hydroxy ketone 12 to
25-[(triethylsilyl)oxy]-des-A,B-18-norcholestan-8-one (13)
A solution of the ketone 12 (5 mg, 19 .mu.mol) and imidazole (15
mg, 220 .mu.mol) in anhydrous DMF (150 .mu.L) was treated with
triethylsilylchloride (15 .mu.L, 90 .mu.mol). The mixture was
stirred at room temperature under argon for 4 h. Ethyl acetate was
added and water, and the organic layer separated. The ethyl acetate
layer was washed with water and brine, dried (MgSO.sub.4), filtered
and evaporated. The residue was passed through a silica Sep-Pak in
10% ethyl acetate in hexane, and after evaporation purified by HPLC
(9.4 mm.times.25 cm Zorbax-Sil column, 4 mL/min) using hexane/ethyl
acetate (9:1) solvent system. Pure protected ketone 13 (3.6 mg,
50%) was eluted at R.sub.v 25 mL as a colorless oil: .sup.1 H NMR
(CDCl.sub.3) .delta. 0.559 (6H, q, J=7.9 Hz, 3.times.SiCH.sub.2),
0.896 (3H, d, J=7.6 Hz, 21-H.sub.3), 0.939 (9H, t, J=7.9 Hz,
3.times.SiCH.sub.2 CH.sub.3), 1.183 (6H, s, 26- and
27-H.sub.3).
EXAMPLE 8
Preparation of 1.alpha.,25-dihydroxy-18,19-dinor-vitamin D.sub.3
(16)
(Scheme II)
[2-[(3R,,5R)
-3,5-Bis[(tert-butyldimethylsilyl)oxy]-cyclohexylidene]ethyl]diphenylphosp
hine oxide (14) (12 mg, 21 .mu.mol) was dissolved in anhydrous THF
(200 .mu.L), cooled to -78.degree. C. and n-BuLi (1.4M in hexanes,
15 .mu.L, 21 .mu.mol) added under argon with stirring. The solution
turned deep orange. After stirring for 5 min at -78.degree. C. the
protected ketone 13 (3.0 mg, 7.9 .mu.mol) was added in anhydrous
THF (200 .mu.L+100 .mu.L). The mixture was stirred under argon at
-78.degree. C. for 1 h and at 0.degree. C. for 16 h. Ethyl acetate
was added and the organic phase washed with saturated NH.sub.4 Cl,
10% NaHCO.sub.3 and brine, dried (MgSO.sub.4) and evaporated. The
residue was passed through a silica Sep-Pak in 10% ethyl acetate in
hexane, and after evaporation purified by HPLC (9.4 mm.times.25 cm
Zorbax-Sil column, 4 mL/min) using hexane-ethyl acetate (9:1)
solvent system. Pure protected vitamin 15 (1.7 mg, 29%) was eluted
as a colorless oil: .sup.1 H NMR (CDCl.sub.3) .delta. 0.045 and
0.054 (6H and 6H, each s, 4.times.SiCH.sub.3), 0.557 (6H, q, J=7.9
Hz, 3.times.SiCH.sub.2), 0.86-0.87 (21H, 21-H.sub.3 and
2.times.Si-t-Bu), 0.939 (9H, t, J=7.9 Hz, 3.times.SiCH.sub.2
CH.sub.3), 1.178 (6H, br s, 26- and 27-H.sub.3), 2.84 (1H, br d,
J=13.5 Hz, 9.beta.-H), 4.07 (2H, br m, 1.beta.- and 3.alpha.-H),
5.90 and 6.14 (1H and 1H, each d, J=11.1 Hz, 7- and 6-H).
Protected vitamin 15 described above (850 .mu.g, 1.2 .mu.mol) was
dissolved in benzene (40 .mu.L) and cation exchange resin (AG
50W-X4, 17 mg; prewashed with methanol) in methanol (200 .mu.L) was
added. The mixture was stirred at room temperature under argon for
18 h, filtered through a silica Sep-Pak and washed with 2-propanol.
The solvent was evaporated and a crude vitamin 16 was purified by
HPLC (10 mm.times.25 cm Zorbax-Sil column, 4 mL/min) using
hexane/2-propanol (7:3) solvent system. Analytically pure compound
16 (366 .mu.g, 81%) was eluted at R.sub.v 37 mL
(1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3 was eluted at R.sub.v
36 mL in the same system) as a white solid: UV (in EtOH)
.lambda..sub.max 243, 251.5, 261 nm; .sup.1 H NMR (CDCl.sub.3)
.delta. 0.879 (3H, d, J=6.5 Hz, 21-H.sub.3), 1.208 (6H, s, 26- and
27-H.sub.3), 4.07 and 4.11 (1H and 1H, each m, 1.beta.- and
3.alpha.-H), 5.94 and 6.30 (1H and 1H, each d, J=11.2 Hz, 7- and
6-H); MS m/z (relative intensity) v; exact mass calcd for C.sub.25
H.sub.42 O.sub.3 390.3134, found 390.3139
v 390(M+,39), 372(62), 354(23), 259(42), 231(84), 175(25), 149(25),
133(53), 121(64), 69(100) ##STR8##
For treatment of bone diseases, the novel compounds of this
invention defined by formula I may be formulated for pharmaceutical
applications as a solution in innocuous solvents, or as an
emulsion, suspension or dispersion in suitable solvents or
carriers, or as pills, tablets or capsules, together with solid
carriers, according to conventional methods known in the art. Any
such formulations may also contain other
pharmaceutically-acceptable and non-toxic excipients such as
stabilizers, anti-oxidants, binders, coloring agents or emulsifying
or taste-modifying agents.
The compounds may be administered orally, parenterally or
transdermally. The compounds are advantageously administered by
injection or by intravenous infusion of suitable sterile solutions,
or in the form of liquid or solid doses via the alimentary canal,
or in the form of creams, ointments, patches, or similar vehicles
suitable for transdermal applications. Doses of from 0.1 .mu.g to
50 .mu.g per day of the compounds are appropriate for treatment
purposes, such doses being adjusted according to the disease to be
treated, its severity and the response of the subject as is well
understood in the art. Since the new compounds exhibit specificity
of action, each may be suitably administered alone, or together
with graded doses of another active vitamin D compound--e.g.
1.alpha.-hydroxyvitamin D.sub.2 or D.sub.3, or
1.alpha.,25-dihydroxyvitamin D.sub.3 --in suitations where
different degrees of bone mineral mobilization and calcium
transport stimulation is found to be advantageous.
Compositions for use in the above-mentioned treatment of psoriasis
and other malignancies comprise an effective amount of one or more
18,19-dinor-vitamin D compound as defined by the above formula I as
the active ingredient, and a suitable carrier. An effective amount
of such compounds for use in accordance with this invention is from
about 0.01 .mu.g to about 100 .mu.g per gm of composition, and may
be administered topically, orally or parenterally in dosages of
from about 0.01 .mu.g/day to about 100 .mu.g/day.
The compounds may be formulated as creams, lotions, ointments,
topical patches, pills, capsules or tablets, or in liquid form as
solutions, emulsions, dispersions, or suspensions in
pharmaceutically innocuous and acceptable solvent or oils, and such
preparations may contain in addition other pharmaceutically
innocuous or beneficial components, such as stabilizers,
antioxidants, emulsifiers, coloring agents, binders or
taste-modifying agents.
The compounds may be administered topically, as oral doses, or
parenterally by injection or infusion of suitable sterile
solutions. The compounds are advantageously administered in amounts
sufficient to effect the differentiation of promyelocytes to normal
macrophages. Dosages as described above are suitable, it being
understood that the amounts given are to be adjusted in accordance
with the severity of the disease, and the condition and response of
the subject as is well understood in the art.
Biological Activity of 18,19-Dinor-Vitamin D Compounds
The 18,19-dinor compounds of this invention exhibit a pattern of
biological activity having high potency in promoting the
differentiation of malignant cells, relatively high intestinal
calcium transport activity and a relatively low ability to mobilize
calcium from bone. This is illustrated by the biological assay
results obtained for 1.alpha.,25-dihydroxy-18,19-dinor-vitamin
D.sub.3 which are summarized in FIGS. 1 and 2 and in Table 1
respectively. FIG. 1 shows a comparison of the activity of the
known active metabolite 1.alpha.,25-dihydroxyvitamin D.sub.3 and
the 19-nor analog 1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3 and
the presently claimed 18,19-dinor-1.alpha.,25-dihydroxyvitamin
D.sub.3 in inducing the differentiation of human leukemia cells
(HL-60 cells) in culture to monocytes. Differentiation activity was
assesed by a standard differentiation assay, abbreviated in FIG. 1
as NBT reduction (nitroblue tetrazolium reduction). The assay was
conducted according to known procedures, as given, for example, by
DeLuca et al U.S. Pat. No. 4,717,721 and Ostrem et al, J. Biol.
Chem. 262, 14164, 1987. For the assay, the differentiation activity
of the test compounds is expressed in terms of the percent of HL-60
cells having differentiated to normal cells in response to a given
concentration of test compound.
The results summarized in FIG. 1 clearly show that the analog,
1.alpha.,25-dihydroxy-18,19-dinor-vitamin D.sub.3 is as potent as
1.alpha.,25-dihydroxyvitamin D.sub.3 in promoting the
differentiation of leukemia cells. Thus in the NBT assay close to
90% of the cells are induced to differentiate by
1.alpha.,25-dihydroxy-vitamin D.sub.3 at a concentration of
1.times.10.sup.-7 M, and the same degree of differentiation is
achieved by the 18,19-dinor analog at 1.times.10.sup.-7 M.
FIG. 2 illustrates the relative activity of
18-nor-1.alpha.,25-dihydroxyvitamin D.sub.3,
19-nor-1.alpha.,25-dihydroxyvitamin D.sub.3,
18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3 and
1.alpha.,25-dihydroxyvitamin D.sub.3 in binding to the
1.alpha.,25-dihydroxyvitamin D pig intestinal nuclear receptor.
FIG. 2 shows that 18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3
is very active in binding to the 1.alpha.,25-dihydroxyvitamin
D.sub.3 receptor from porcine intestinal nuclei.
Table 1 shows a comparison of the calcemic activity of the known
active metabolite 1.alpha.,25-dihydroxyvitamin D.sub.3, and the
19-nor analog 1.alpha.,25-dihydroxy-19-nor-vitamin D.sub.3 and the
presently claimed 18,19-dinor-1.alpha.,25-dihydroxyvitamin
D.sub.3.
TABLE 1 ______________________________________ INTESTINAL CALCIUM
TRANSPORT AND BONE CALCIUM MOBILIZING ACTIVITIES OF
1.alpha.,25-DIHYDROXY- VITAMIN D.sub.3 COMPOUNDS Serum Ca Dosage
S/M (Ave. .+-. SEM) Compound (Pmoles) (Ave. .+-. SEM) (mg/100 ml)
______________________________________ D deficient 0 5.02 .+-. 0.22
4.83 .+-. 0.1 1,25(OH).sub.2 D.sub.3 1,000 13.5 .+-. 0.89 7.15 .+-.
0.24 19-Nor-1,25- 1,000 10.4 .+-. 0.40 5.10 .+-. 0.14 (OH).sub.2
D.sub.3 18,19-Dinor- 1,000 10.4 .+-. 0.85 5.66 .+-. 0.07 1,25-
(OH).sub.2 D.sub.3 ______________________________________
Male, weanling rats (Sprague-Dawley) were fed a low calcium vitamin
D-deficient diet for three weeks and then received the indicated
dose dissolved in 95% propylene glycol/5% ethanol
intraperitoneally. 24 hours later, blood serum was obtained, and
calcium determined in the presence of 0.1% lanthanum chloride,
using an atomic absorption spectrometer. The control animals
received the vehicle alone. The values are the mean.+-.standard
error of the mean. There were at least 6 animals per group.
Table 1 shows that 18,19-dinor-1.alpha.,25-dihydroxyvitamin
D.sub.3, while having some ability to mobilize calcium from bone,
is clearly not as active in this regard as
1.alpha.,25-dihydroxyvitamin D.sub.3. Also, Table 1 shows that
18,19-dinor-1.alpha.,25-dihydroxyvitamin D.sub.3 is almost as
active as 1.alpha.,25-dihydroxyvitamin D.sub.3 in intestinal
calcium transport activity.
Thus, the 18,19-dinor analog shows a selective activity profile
combining high potency in inducing the differentiation of malignant
cells, relatively high intestinal calcium transport activity with
relatively low bone mobilization activity. The compounds of this
novel structural class, therefore, can be useful as therapeutic
agents for the treatment of psoriasis and other malignancies, and
for the treatment of metabolic bone diseases where bone loss is a
major concern such as osteoporosis, osteomalacia and renal
osteodystrophy.
Various modes of carry out the invention are contemplated as being
within the scope of the following claims, particularly pointing out
and distinctly claiming the subject matter regarded as the
invention.
* * * * *